Infections diseases have plagued life on earth probably from its inception. Such diseases affect not only man but animals. In economically advanced countries of the world, infection diseases are 1) temporarily disabling; 2) permanently disabling or crippling; or 3) fatal. In the lesser developed countries, infections diseases tend to fall into the latter two categories, permanently disabling or crippling and fatal, due to may factors, including a lack of preventive immunization and curative medicine.
It is generally acknowledged that the usefulness of antibiotics to effectively control bacterial pathogens is becoming increasingly difficult, because of the increased occurrence of antibiotic-resistant pathogens. Thus, prevention of infectious diseases is more cost effective than the ultimate treatment of the disease once it has occurred. As a result, increased attention is being focused on the development of vaccines.
Vaccines are administered to humans and animals to induce their immune systems to produce antibodies against viruses, bacteria, and other types of pathogenic organisms. In the economically advanced countries of the world, vaccines have brought many diseases under control. In particular, many viral diseases are now prevented due to the development of immunization programs. The virtual elimination of smallpox is an example of the effectiveness of a vaccine worldwide. But many vaccines for such diseases as poliomyelitis, measles, mumps, rabies, foot and mouth, and hepatitis B are still too expensive for the lesser developed countries to provide to their large human and animal populations. Lack of these preventative measures for animal populations can worsen the human condition by creating food shortages.
Because of simplicity of delivery of vaccines by oral delivery, there is great current interest in discovering new oral vaccine technology. Appropriately delivered oral immunogens can stimulate both humoral and cellular immunity and have the potential to provide cost-effective, safe vaccines for use in developing countries or inner cities where large-scale parenteral immunization is not practical or extremely difficult to implement. Such vaccines may be based upon bacterial or viral vector systems expressing protective epitopes from diverse pathogens (multivalent vaccines) or may be based upon purified antigens delivered singularly or in combination with relevant antigens or other pathogens.
A. Microbial Pathogenesis and Oral Vaccination
Microbial pathogens can infect a host by one of several mechanisms. They may enter through a break in the integument induced by trauma, they may be introduced by vector transmission, or they may interact with a mucosal surface. The majority of human pathogens initiate disease by the last mechanism, i.e., following interaction with mucosal surfaces. Bacterial and viral pathogens that act through this mechanism first make contact with the mucosal surface where they may attache and then colonize, or be taken up by specialized absorptive cells (M cells) in the epithelium that overly Peyer's patches and other lymphoid follicles. Organisms that enter the lymphoid tissues may be readily killed within the lymphoid follicles, thereby provoking a potentially protective immunological response as antigens are delivered to immune cells within the follicles (e.g., Vibrio cholerae). Alternatively, pathogenic organisms capable of surviving local defense mechanisms may spread from the follicles and subsequently cause local or systemic disease (e.g., Salmonella spp., poliovirus in immunocompromised hosts).
Secretory IgA (sIgA) antibodies directed against specific virulence determinants of infecting organism play an important role in overall mucosal immunity. In many cases, it is possible to prevent the initial infection of mucosal surfaces by stimulating production of mucosal sIgA levels directed against relevant virulence determinants of an infecting organism. Secretory IgA may prevent the initial interaction of the pathogen with the mucosal surface by blocking attachment and/or colonization, neutralizing surface acting toxins, or preventing invasion of the host cells.
Parenterally administered inactivated whole-cell and whole-virus preparations are effective at eliciting protective serum IgG and delayed type hypersensitivity reactions against organisms that have a significant serum phase in their pathogenesis (e.g., Salmonella typhi, Hepatitis B). However, parenteral vaccines are not effective at eliciting mucosal sIgA responses and are ineffective against bacteria that interact with mucosal surfaces and do not invade (e.g., Vibrio cholerae).
Oral immunization can be effective for induction of specific sIgA responses if the antigens are presented to the T and B lymphocytes and accessory cells contained within the Peyer's patches where preferential IgA B-cell development is inititated. The Peyer's patches contain helper T cells (TH) that mediate B-cell isotype switching directly from IgM cells to IgA B cells then migrate to the mesentric lymph nodes and undergo differentiation, enter the thoracic duct, then the general circulation, and subsequently seed all of the secretory tissues of the body, including the lamina propria of the gut and respiratory tract. IgA is then produced by the mature plasma cells, complexed with membrane-bound Secretory Component, and transported onto the mucosal surface where it is available to interact with invading pathogens. The existence of this common mucosal immune system explains in part the potential of live oral vaccines and oral immunization for protection against pathogenic organisms that initiate infection by first interacting with mucosal surfaces.
A number of strategies have been developed for oral immunization, including the use of attenuated mutants of bacteria (e.g., Salmonella spp.) as carriers of heterologous antigens, encapsulation of antigens into microspheres composed of poly-DL-lactide-glycolide (PGL), protein-like polymers-proteinoids, gelatin capsules, different formulations of liposomes, adsorption onto nanoparticles, use of lipophilic immune stimulating complexes, and addition of bacterial products with known adjuvant properties.
Underlying the development of most current vaccines is the ability to grow the disease causing agent in large quantities. At present, vaccines are usually produced from killed or live attenuated pathogens. If the pathogen is a virus, large amounts of the virus must be grown in an animal host or cultured animal cells. If a live attenuated virus is utilized, it must be clearly proven to lack virulence while retaining the ability to establish infection and induce humoral and cellular immunity. If a killed virus is utilized, the vaccine must demonstrate the lack of capacity of surviving antigens to induce immunization. Additionally, surface antigens, the major viral particles which induce immunity, may be isolated and administered to induce immunity in lieu of utilizing live attenuated or killed viruses.
Enteric bacterial diseases such as cholera, dysentery, Escherichia coil (E. coli) related diarrheas, and typhoid fever are major causes of morbidity and mortality worldwide, especially in developing countries where sanitation conditions are often less than adequate. Several types of vaccines against these enteropathies have been developed and tested over the years, among them killed whole cells, subunits of toxins, and live attenuated bacteria administered parenterally or orally. The majority of the morbidity and mortality due to bacterial diarrheal disease results from infections with V. cholerae and the cholera-related enterotoxic enteropathies (viz., E. coli that produce cholera-like enterotoxin).
E. coli cause diarrheal disease by a variety of mechanisms, including production of one or more enterotoxins. One of these toxins, referred to as the heat-labile enterotoxin (LT), is immunologically and physiochemically related to the cholera enterotoxin. LT has been purified to homogeneity and has been extensively characterized. LT-B, a 56,000 dalton pentameric protein that consists of 5 identical monomeric subunits, functions as the binding component of the heat-labile enterotoxin. If administered parenterally, LT-B induces a serum response to itself and to haptens covalently linked to the molecule even in the absence of an adjuvant. Likewise, LT-B administered orally has been shown in both animal models and in human volunteers, to induce a serum IgG and mucosal IgA response to itself and to appropriately linked haptens.
The structure of LT has been well characterized by X-ray crystallography and consists of multimeric protein. The holotoxin includes one `A` subunit (LT-A) of molecular weight 27,000 Daltons which is cleaved into LT-A1 and LT-A2 by the proteases in the small bowel. The holotoxin also contains five `B` subunits (LT-B), of molecular weight 11,600 Daltons each, that are noncovalently linked into a very stable doughnutlike pentamer structure.
The LT-B pentamer structure binds to intestinal epithelial cells via specific interactions with the GM-I ganglioside (galactosyl-N-acetylgalactosaminyl-(sialyl)-galactosylglucosyl ceramide) present on the cell surface. This facilitates the entry of toxic LT-A1 into cells, which contains the ADP ribosyl transferase activity. The LT toxin of enterotoxigenic E. coli (ETEC) is similar both structurally and functionally to CT, the cholera toxin of Vibrio cholera. Immunization against one has been seen to lead to cross protection against the other. J. D. Clemens, et al., Lancet 335, 270 (1990). The B subunit of cholera is an integral part of the candidate oral vaccine tested in Bangladesh. J. D. Clemens, et. al., Lancet 335, 270 (1990).
Vaccine manufacturing often employs complex technologies entailing high costs for both the development and production of the vaccine. Concentration and purification of the vaccine is required, whether it is made from cell cultures, whole bacteria, viruses, other pathogenic organisms or sub-units thereof. The high cost of purifying a vaccine in accordance with Food and Drug Administration (FDA) regulations makes most oral vaccines prohibitively expensive to produce because they require ten to fifty times more than the regular quality of vaccine per dose than a vaccine which is parenterally administered.
According to FDA guidelines, efficacy of vaccines for humans must be demonstrated in animals by antibody development and by resistance to infection and disease upon challenge with the pathogen. When the safety and immunogenicity level are satisfactory, FDA clinical studies are then conducted in humans. A small carefully controlled group of volunteers are enlisted from the general population to begin human trials. This long and expensive process of testing takes years before it can be determined whether the vaccine can be given to the general population. If the trials are successful, the vaccine may then be mass produced and sold to the public.
Even after these precautions, problems can and do arise. With killed bacterial cells, viruses or other pathogenic organisms, there is always a chance that live pathogens survive and vaccination may lead to isolated cases of the disease. Moreover, the vaccines may sometimes be contaminated with cellular material from the culture material from which it was derived. These contaminates can cause adverse reactions in the vaccine recipient and sometimes even death. Legal liability of the vaccine manufacturer for those who are harmed by an adverse reaction to vaccines requires the manufacturer to have and maintain expensive liability insurance which further adds to the ultimate cost of the vaccine.
Most pathogens enter on or through a mucosal surface, with exception of insect-borne pathogens or pathogens entering the body through wounds. Pathogens that enter, through mucosal surfaces include, without limitation, Actinomyces, Aeromonas, Bacillus, Bacteroides, Bordetella, Brucella, Compylobacter, Capnbocylophaga, Clamydia, Clostridium, Corynebacterium, Eikenella, Erysipelothriz, Escherichia, Fusobacterium, Hemophilus, Klebsiella, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Nocardia, Pasteurella, Proteus, Pseudomonas, Rickettsia, Salmonella, Selenomonas, Shigelia, Staphylococcus, Streptococcus, Treponema, Bibro, and Yersinia, pathogenic viral strains from the groups Adeiiovirus, Coronavirus, Herpesvirus, Orthomyxovirus, Picornovirus, Poxvirus, Reovirus, Retrovirus, and Rotavirus, pathogen fungi from the general Aspercillus, Blastomyces, Candida, Coccoidiodes, Cryptococcus Histoplasma and Phycomyces, and pathogenic parasites in the general Eimeria, Entamoeba, and Trichomonas.
Mammalian hosts infected by a pathogen mount an immune response in an attempt to overcome the pathogen. The immune system consists of three branches: mucosal, humoral, and cellular. Mucosal immunity results from the production of secretory (sIgA) antibodies in secretions that bathe all mucosal surfaces including the respiratory tract, gastrointestinal tract, and the genitourinary tract and in secretions from all secretory glands. Secretory IgA antibodies prevent colonization of pathogens on the mucosal surfaces and are a first line of defense against colonization and invasion of a pathogen through the mucosal surfaces. The production of sIgA can be stimulated wither by local immunization of the secretory gland or tissue or by presentation of an antigen to either the gut-associated lymphoid tissue (GALT or Peyer's patches) or the bronchial-associated lymphoid tissue (BALT).
Membranous microfold cells, otherwise known as M cells, cover the surface of the GALT and BALT and may be associated with other secretory mucosal surfaces. M cells act to sample antigens from the luminal space adjacent to the mucosal surface and transfer such antigens to antigen-presenting cells (dendritic cells and macrophages), which in turn present the antigen to T lymphocytes (in the case of T-dependent antigens), which process the antigen for presentation to committed B cells. B cells are then stimulated to proliferate, migrate, and ultimately transformed into antibody-secreting plasma cells producing IgA against the presented antigen.
When the antigen is taken up by M cells overlying the GALT and BALT, a generalized mucosal immunity results with sIgA against the antigen being produced by all secretory tissues in the body. Because most pathogens enter through mucosal surfaces and such surfaces make up the first line of defense to infection and facilitate the body's immune response, vaccines that can be orally administered represent a most important route to stimulating a generalized mucosal immune response leading to local stimulation of a secretory immune response in the oral cavity and in the gastrointestinal tract.
Secretory IgA antibodies directly inhibit the adherence of microorganisms to mucosal epithelial cells and to the teeth of the host. This inhibition may be the result of agglutination of microorganisms, reduction of hydrophobicity or negative charge, and blockage of microbial adhesions. These anti-adherence effects are amplified by other factors such as secretory glycoproteins, continuous desquamation of surface epithelium and floral competition.
Clinical experience with human peroral poliovirus vaccine and several peroral or intranasal virus vaccines applied in veterinary medicine shown that sIgA plays a decisive role in the protective effect by the mucosal immune system against respiratory and enteric viral infections. The effect of sIgA appears to be that of inhibiting the entry of viruses into host cells rather than prevention of attachment.
B. Plant Genetic Engineering
Various methods are known in the art to accomplish the genetic transformation of plants and plant tissues so that foreign DNA is introduced into the plant's genetic material in a stable manner, i.e., a manner that will allow the foreign DNA to be passed on the plant's progeny. Two such transforming procedures are Agrobacterium-mediated transformation and direct gene transfer.
Agrobacterium-mediated transformation utilizes A. tumefaciens, the etiologic agent of crown gall, a disease of a wide range of dicotyledons and gymnosperms that results in the formation of tumors or galls in plant tissue at the site of infection. Agrobacterium, which normally infects the plant at wound sites, carries a large extrachromosomal element called Ti (tumor-inducing) plasmid.
Ti plasmids contain two regions required for tumor induction. One region is the T-DNA (transferred-DNA) which is the DNA sequence that is ultimately found stably transferred to plant genomic DNA. The other region is the vir (virulence) region which has been implicated in the transfer mechanism. Although the vir region is absolutely required for stable transforma-tion, the vir DNA is not actually transferred to the infected plant. Transformation of plant cells mediated by infection with A. tumefaciens and subsequent transfer of the T-DNA alone have been well documented. See, e.g., Bevan, M. W. et al., Int. Rev. Genet, 16, 357 (1982).
After several years of intense research in many laboratories, the Agrobacterium system has been developed to permit routine transformation of a variety of plant tissues. Representative tissues transformed by this technique include, but are not limited to, tobacco, tomato, sunflower, cotton, rapeseed, potato, poplar, and soybean.
A. rhizogenes has also been used as a vector for plant transformation. That bacterium, which incites root hair formation in many dicotyledonous plant species, carries a large extrachromosomal element called a Ri (root-inducing) plasmid which functions in a manner analogous to the Ti plasmid of A. tumefaciens. Transformation using A. rhizogenes has developed analogously to that of A. tumefaciens and has been successfully utilized to transform plants which include but are not limited to alfalfa and poplar.
In the case of direct gene transfer, foreign genetic material is transformed into plant tissue without the use of the agrobacterium plasmids. Direct transformation involves the uptake of exogenous genetic material into plant cells or protoplasts. Such uptake may be enhanced by use of chemical agents or electric fields. The exogenous material may then be integrated into the nuclear genome. The early work with direct transfer was conducted in the dicot Nicotiana tobacum (tobaco) where it was shown that the foreign DNA was incorporated and transmitted to progeny plants. Several monocot protoplasts have also been transformed by this procedure including maize and rice.
Liposome fusion has also been shown to be a method for transforming plant cells. Protoplasts are brought together with liposomes carrying the desired gene. As membranes merge, the foreign gene is transferred to the protoplast.
In addition, direct gene transfer can be accomplished by polyethylene glycol (PEG) mediated transformation. PEG mediated transformation has been successfully used to transform dicots such as tobacco and monocots such as lolium multiflorum. This method relies on chemicals to mediate the DNA uptake by protoplasts and is based on synergistic interactions between MG.sup.+2, PEG, and possibly Ca.sup.+2. See, e.g., Negrutiu, R. et al., Plant Mol. Biol., 8, 363 (1987).
Alternatively, exogenous DNA can be introduced into cells or protoplasts by microinjection. In this technique, a solution of the plasmid DNA or DNA fragment is injected directly into the cell with a finely pulled glass needle. This technique has been used to transform alfalfa.
A more recently developed procedure for direct gene transfer involves bombardment of cells by micro-projectiles carrying DNA. In this procedure, commonly called particle bombardment, tungsten or gold particles coated with the exogenous DNA are accelerated toward the target cells. The particles penetrate the cells carrying withthem the coated DNA. Microparticle acceleration has been successfully demonstrated to leas to both transient expression and stable expression in cells suspended in cultures, protoplasts, immature embryos of plants including but not limited to onion, maize, soybean, and tobacco.
Once plant cells have been transformed, there are a variety of methods for regenerating plants. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated. In recent years, it has become possible to regenerate many species of plants from callus tissue derived from plant explants. The plants which can be regenerated from callus include monocots, such as but not limited to corn, rice, barley, wheat, and rye, and dicots, such as but not limited to sunflower, soybean, cotton, rapeseed and tobacco.
Regeneration of plants from tissue transformed with A. tumefaciens has been demonstrated for several species of plants. These include but is not limited to sunflower, tomato, white clover, rapeseed, cotton, tobacco, potato, maize, rice, and numerous vegetable crops.
Plant regeneration from protoplasts is occasionally a useful technique. When a plant species can be regenerated from protoplasts, then direct gene transfer procedures can be utilized, and transformation is not dependent on the use of A. tumefaciens. Regeneration of plants from protoplasts has been demonstrated for plants including but not limited to tobacco, potato, poplar, corn, and soybean.
The technology developed for the creation of transgenic plants has led many investigators to study the expression of genes derived from dissimilar plant species or from non-plant genomes. In many cases, it has been desirable to characterize the expression of recombinant proteins encoded by genes derived from viruses or bacteria. The construction of chimeric genes for expression of foreign coding sequences in plants involves ligation of non-coding regulatory elements which function in plants 5' to the DNA sequence encoding the desire protein, and ligation of a polyadenylation signal which is active in plant cells 3' to the DNA sequence encoding the desired protein.
The 5' regulatory sequences which are often used in creation of chimeric genes for plant transformation may cause either nominally constitutive expression in all cells of the transgenic plant, or regulated gene expression where only specific cells or tissues show expression of the introduced genes. The CaMV 35-S promoter, which was derived from the Cauliflower Mosaic Virus which causes a plant disease, has frequently been used to drive nominally constitutive expression of foreign genes in plants. A regulatory DNA element which was found to control the tuber-specific expression of the patatin protein is an example of developmentally specific gene expression; this patatin promoter element is known to cause the tuber-specific expression of at least some foreign genes. See, e.g., H. C. Wenzler, G. A. Mignery, L. M. Fisher, W. D. Park, Plant Mol. Biol., 12:41-50 (1989).
Chimeric gene constructions may also include modifications of the amino acid coding sequence of the structural gene being introduced into transgenic plants. For example, it may be desirable to add or delete amino acids in the protein to be expressed to influence the cellular localization of foreign gene product in the cells of transgenic plants.
It has been shown that the inclusion of KDEL and HDEL amino acid sequences at the carboxy terminus of at least one protein enhanced the recognition for that protein by the plant endoplasmic reticulum retention machinery. S. Munro and H. R. b. Pelham, Cell 48, 988-997 (1987); J. Denecke, R. DeRycke, J. Botterman, EMBO-J. 11, 2345 (1992); E. M. Herman, B. W. Tague, L. M. Hoffman, S. E. Kjemtrip, J. J. Chrispeels, Planta 182, 305 (1991); C. Wandelt, et al., The Plant Journal 2, 181 (1992). However, such modifications are problematic at best becasue other factors such as protein conformation or protein folding in the transformed cells may interfere with the availability of this carboxy terminus signal by the plant endoplasmic reticulum retention machinery. S. M. Haugejorden, M. Srinivasan, M. Gree, J-Biol-Chem. 266, 6015 (1991).
C. Oral Vaccine Methodologies Using Transgenic Plants
There are four well-known genetic expression transformation systems that can be used for producing transgenic plants capable of being administered as one of the active agents in oral vaccines against a desired antigen. The present invention takes advantage of all four of these expression systems of the construction of edible vaccines. It should be recognized that this list is not meant to exhaust other possible approaches. The list is simply included to provide a proper context for the scope and teaching of the present invention.
First, in transgenic plants, expression vectors including the CaMV 35S promoter and antigen coding sequences can be used to constitutively transform the plants where expression in the leaves allows for rapid analysis of gene expression and biochemical characterization of gene products.
In such plants such as but not limited to Brassica napus (canola), expression vectors including the 2S albumin promoter and antigen coding sequences can be used to cause seed-specific gene expression to create the production of recombinant protein in seed tissues, routinely used as animal feed, providing for the production of attractive oral immunogenicity analyses.
In plants such as but not limited to Solanum tuberosum (potato), expression vectors including the patatin promoter or soybean vspB promoter and antigen coding sequences can be used to cause tuber-specific gene expression to create tuber-specific production of recombinant protein in tuber tissues routinely used as food. This provides for the production of attractive oral immunogenicity analyses.
Finally, in plants such as but not limited to Musa acuminata (banana), expression vectors including fruit ripening-specific promoters and antigen coding sequences can be used to transform plants that produce the recombinant protein in ripened fruit where production of recombinant protein is produced directly as candidate vaccines for ingestion studies in animals and humans.
The retention of biological properties in the recombinant proteins produced in plants, specifically ligand binding and the presentation of antigenic epitopes, is of considerable importance to the successful production of edible vaccines in transgenic plants. The ultimate test of the value of proteins of pharmacological importance is their biological activity. Vaccines are of particular interest for studies of protein expression since their effects can be accurately quantified in animal models. In addition, relatively low amounts are required, since their effects are amplified by the immune system.
The high cost of production and purification of synthetic peptides manufactured by chemical or fermentation based processes may prevent their broad scale use as oral vaccines. The production of immunogenic proteins in transgenic plants and the adjuvant effect of such proteins in transgenic plants offers an economical alternative.
While oral vaccines may be an effective and inexpensive procedure for inducing secretory immune responses in animals including humans, there is a need for proven techniques that yield transgenic plants or plant tissue that can, upon direct ingestion, cause a desired immune response to a given antigen without significant side effects.
Attempts to produce transgenic plants expressing bacterial antigens of E. coli and of Streptococcus mutans have been made. Curtiss and Ihnen, WO 90/0248, published Mar. 22, 1990. Transgenic plants which express the Hepatitis B surface antigen (HBsAg) have also been made. H. S. Mason, D. M-K. Lam, C. J. Arntzen, Proc. Nat. Acad. Sci. USA, 89:11745-749 (1992).
However, those studies have not yielded orally immunogenic plant material nor have they demonstrated that it is, in fact, possible to orally immunize animals with antigens produced in transgenic plants. In fact, it took the significant and unexpected improvements disclosed herein, to successfully demonstrate that it is indeed possible to actually immunize animals against antigens by feeding animals transgenic plants in which sufficient levels of the antigen have been expressed in order to induce immunity. In addition, it took the significant and unexpected improvements of the instant invention to also demonstrate the adjuvant effect toward other immunogens caused by immunizing animals with transgenic plants containing bacterial toxin antigens.
The present discovery described in the following sections discloses how to overcome the previous limitations by the significant and unexpected improvements of causing the production of increased levels of the antigenic protein and by compartmentalization in microsomal vesicles of transgenic plants of an orally active LT-B protein which is highly immunogenic, and by the increased expression of such transgenic bacterial antigens in plant through i) alterations in the plant promoters to increase the level of expression of transgenic proteins in plants, ii) alteration of 3' messages and polyadenylation signals to increase the production of transgenic proteins in plants and iii) production of synthetic genes encoding bacterial antigens where the codon usage has been changed to increase the usage by plant thus increasing the level of transgenic proteins produced in plants. Further, the present discovery discloses that the compartmentalized foreign protein expression in edible transgenic plant tissues allows expression and delivery of additional, desired antigens of value as oral vaccines, sine the microsome-encapsulated LT-B serves as an oral adjuvant.